In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

Summary

Selectively labeled fluorescent GnRH-I, -II and -III derivatives are reliable tools for tracking and quantifying their cellular uptake. This manuscript introduces experiments to visualize, quantify and compare the uptake efficiency of these GnRH conjugates in various cell lines.

Abstract

GnRH analogues are effective targeting moieties and able to deliver anticancer agents selectively into malignant tumor cells which highly express GnRH receptors. However, the quantitative analysis of GnRH analogues' cellular uptake and the investigated cell types in GnRH-based drug delivery systems are currently limited. Previously introduced, selectively labeled fluorescent GnRH I, -II and -III derivatives provide great detectability, and they have suitable chemical properties for reproducible and robust experiments. We also found that the appropriate up-to-date methods with these labeled GnRH analogues could offer novel information about the GnRH-based drug delivery systems. This manuscript introduces some simple and fast experiments regarding the cellular uptake of [D-Lys⁶(FITC)]-GnRH-I, [D-Lys⁶(FITC)]-GnRH-II and [Lys⁸(FITC)]-GnRH-III on the EBC-1 (lung), the BxPC-3 (pancreas) and on the Detroit-562- (pharynx) malignant tumor cells. In parallel with these GnRH-FITC conjugates, the cell surface level of GnRH-I receptors was also examined on these cell lines before and after the GnRH treatment by confocal laser scanning microscopy. The cellular uptake of GnRH-FITC conjugates was quantified by fluorescence-activated cell sorting. In these experiments minor differences among GnRH analogues and major differences among cell types was observed. The significant differences among cell lines are correlated with their distinct level of cell surface GnRH-I receptors. The introduced experiments contain practical methods to visualize, quantify and compare the uptake efficiency of GnRH-FITC conjugates in a time- and concentration-dependent manner on various adherent cell cultures. These results could predict the drug targeting efficiency of GnRH conjugates on the given cell culture, and offer a good basis for further experiments in the examination of GnRH-based drug delivery systems.

Introduction

Peptide based targeted drug delivery systems have become a fast developing and promising area in cancer therapy over the past few years^1,2. Human gonadotropin releasing hormone receptor type I (GnRH-I-R) is primarily located in the pituitary gland but is also present in several other tissues which are responsible for self-reproduction³. GnRH-I-R is also expressed in a number of cancer tissues, related or unrelated to the reproductive system^4,5. The high expression of GnRH-I-R on several malignant tumor cells compared with health....

Protocol

1. Preparation of Cell Cultures and Reagents

1. Maintain the cell cultures in the manufacturer's recommended medium, supplemented with 10% (v/v) fetal bovine serum and antibiotics (called complete medium). Keep the cell culturing flask in a humidified, 5% CO₂ atmosphere incubator at 37 °C. Follow the proliferation and confluency of cells by inverted microscope (using 10X phase contrast objective).
2. When cells reach adequate confluency, remove the medium, and wash the culture with 2-3 mL, sterile phosphate-buffered saline (PBS). Remove the PBS and add 0.5 mL, sterile 0.25% trypsin-EDTA solution to the cell culture and incubate at 3....

Results

Images obtained by confocal laser scanning microscopy (CLSM) offer spectacular information about the uptake of GnRH-FITC conjugates on the given cell culture in a time and concentration dependent manner. In parallel with these GnRH-FITC conjugates, the presence of the GnRH-I-R on the cell surface is also verifiable by the CLSM experiment. Furthermore, by using a far-red DNA staining fluorescent probe, it is possible to counterstain the nuclei of cells besides GnRH and GnRH-I-R. However, w.......

Discussion

Experiments described herein use selectively labeled GnRH analogues for screening adherent cell cultures in vitro. Confocal microscopy and flow cytometry methods are suitable for tracking and quantifying the cellular uptake of these GnRH-FITC conjugates in a time and concentration dependent manner. These experiments have the following critical steps: 1) maintain a sterile, healthy cell culture; 2) GnRH peptides must be of high quality; 3) reasonable concentrations and incubation times must be followed; .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
EBC-1	JCRB Cell Bank	JCRB0820	Human lung squamous cell carcinoma
BxPC-3	ATCC	CRL-1687	Human pancreatic adenocarcinoma
Detroit-562	ATCC	CCL-138	Human pharyngeal carcinoma
RPMI-1640 Medium with L-glutamine	Lonza	BE12-702F	Culture medium for BxPC-3 cells
Eagle's Minimum Essential Medium	Lonza	BE12-611F	Culture medium for EBC-1 cells, and complemented with 0.1% sodium pyruvate for Detroit-562 cells
Fetal Bovine Serum	Euro Clone	ECS0180L	Complements the culture medium
MycoZap Plus-CL	Lonza	VZA-2012	Complements the culture medium (antibiotics)
Standard Line Cell Culture Flasks	VWR	10062-872	Provide consistent, sterile growth environment for cells
Trypsin-EDTA Mixture	Lonza	BE17-161E	Remove attached cells
Phosphate Buffered Saline (PBS)	Lonza	BE17-516F	Solvent
Dimethyl sulfoxide	Sigma-Aldrich	D8418-100ML	Solvent
Trypan Blue Solution, 0.4%	Thermo Fisher Scientific	15250061	Used to counting cells
[D-Lys6(FITC)]-GnRH-I	made by us	-	Purity ≥98% (HPLC)
[D-Lys6(FITC)]-GnRH-II	made by us	-	Purity ≥98% (HPLC)
[Lys8(FITC)]-GnRH-III	made by us	-	Purity ≥98% (HPLC)
glass bottom 8-well microscopic slide	Ibidi	80826	Use in CLSM experiment
Corning Costar cell culture plate, 12 well	Sigma-Aldrich	CLS3513-50EA	Use in FACS experiment
Fixing solution (10% neutral buffered formalin)	Bio-Optica	01V60P	Fix adherent cells
Bovine Serum Albumin	Sigma-Aldrich	A7906-10G	Prevent the nonspecific binding of the antibodies
GnRHR Antibody	Proteintech	19950-1-AP	Immunocytochemistry reagent, bind to the human type-I GnRH receptors
Secondary Antibody, Alexa Fluor 546 conjugate	Thermo Fisher Scientific	A11035	Immunocytochemistry reagent, bind to the primary antibody
Fluorescent probe solution (Draq5)	Thermo Fisher Scientific	62254	Counterstain nuclei
Fluoromount Aqueous Mounting Medium	Sigma-Aldrich	F4680-25ML	Use in CLSM experiment, preserving fluorescence
Inverted microscope	Elektro-Optika Ltd.	Alpha XDS-2T	Check the phenotype and confluency of cell cultures
Inverted confocal laser scanning microscope	Zeiss	LSM-710	Use in CLSM experiment
Flow cytometer	BD Biosciences	BD FACSCalibur	Use in FACS experiment